The invention relates to means for detecting and treating
cancer cells resistant to therapeutic agents, more
particularly to receptor tyrosine kinase (RTK in short)
inhibitors.
Members of the RTK family play an important role in the
proliferation of cancer cells. Aberrant signaling through the
epidermal growth factor receptor (EGFR), a member of the ErbB
family of RTKs, is associated with neoplastic cell
proliferation, migration, invasion, resistance to apoptosis
and angiogenesis.
Upon ligand binding, the EGFR dimerizes, and undergoes
autophosphorylation at specific tyrosine residues of the
intracellular domain. The phosphorylated tyrosines then serve
as docking sites for proteins which, in turn, activate
downstream signaling pathways, including the Ras/MEK/Erk and
the PI3K/Akt pathway, which regulate transcription factors and
other proteins involved in the above-mentioned biological
responses.
Beside cell proliferation, the balance between cell survival
and cell death is a determinant factor in the growth of tumors
and their response to therapeutic agents. It is known that
cell proliferation and apoptosis are controlled by highly
regulated mitogen-activated protein (MAP) kinases, including
ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal
protein kinase), and p38.
MAP kinases participate in signal transduction pathways
through which cells respond functionally to external messages
or to extracellular stresses. Different cell stimuli
preferentially activate distinct MAP kinases. Hence, growth
factors (such as EGF) and oncogenes are linked to activation
of ERK, whereas inflammatory cytokines, growth factor
deprivation and a number of cell stresses lead preferentially
to activation of JNK and p38, which are also called stress-activated
protein kinases (SAPK). The different actors
involved in cell death, survival and proliferation constitute
a network that is highly regulated in normal cells, and
variably altered in cancer cells.
Experimental studies suggest that ERK MAP kinases are involved
in cell cycle progression and mitogenesis, oncogenic
transformation and metastasis, differenciation and survival,
whereas JNK and p38 participate in signaling pathways leading
to apoptosis. How tumor cells regulate the decision point
between either proliferation or survival and cell death is
context-dependent and results from a complex relationship
between the Akt and Erk survival pathways and the SAPK death
pathways.
In particular, several studies indicated that concomitant
inhibition of the ERK or PI3K/Akt signaling pathways with
activation of the SAPK pathways can switch the equilibrium
toward apoptosis in various experimental tumor systems.
Temporal activation of MAP kinases is regulated positively by
upstream kinases and negatively by several classes of
phosphatases, including the serine/threonine phosphatase PP2A,
over 50 tyrosine-specific phosphatases (PTPs) and a growing
family of a subclass of PTPs that possess activity for
dephosphorylating both phosphotyrosine and phosphothreonine
residues, termed dual specificity phosphatases (DSPs).
Tight control of DSP gene induction, combined with their
differential binding and catalytic activation by a specific
repertoire of MAP kinases, provides a sophisticated mechanism
for rapid and targeted inactivation of selected MAP kinase
activities. Hence, the DSPs MKP-3 and M3/6 are highly specific
for inactivating ERK1/2 and either JNK or p38 respectively,
while CL100/MKP-1 and MKP-5 act preferentially on JNK and p38.
Several recent studies suggest that MKP-1 is involved in the
progression of human cancer by protecting tumor cells against
apoptosis. First, MKP-1 expression is induced by low oxygen
conditions found in solid tumor microenvironments, which may
represent a mechanism to protect tumor cells from apoptosis
induced by the activation of JNK in response to hypoxic
stress. Second, MKP-1 expression is increased in several tumor
types, including breast, prostate and ovarian carcinomas, and
was found associated with cell survival in prostate tumor
samples obtained from patients treated by androgen
deprivation.
The high frequency of abnormalities in RTK signaling,
particularly EGFR, in human tumors and laboratory studies
showing that inhibition of EGFR can impair tumor growth, means
that EGFR is an attractive target for the development of
cancer therapeutics.
High expression of the EGFR and/or its ligands is common in
several tumor types, including non-small cell lung carcinoma
(NSCLC), gliomas, head and neck cancer (HNSCC), breast cancer,
and ovarian cancer, and correlates with more aggressive
disease and resistance to chemotherapy.
Several inhibitors of the EGFR and other RTKs are in
development. Gefitinib (ZD1839, Iressa) is an orally
administered, selective and reversible inhibitor of EGFR that
competitively inhibits the binding of ATP required for
receptor autophosphorylation and kinase activation.
Preclinical studies suggest that gefitinib and other EGFR-targeting
agents inhibit tumor cell proliferation and
angiogenesis, induce apoptosis, and show additive or
synergistic cytotoxic effects on tumor cells when used in
combination with standard cancer therapies.
NSCLC is the most frequent cancer in the world and one of the
most lethal as well, with one million deaths worldwide in
2000. Standard chemotherapy for advanced and metastatic NSCLC
is based on various cytotoxic drug combinations. Although
chemotherapy results in a modest survival benefit, four
different reference chemotherapeutic combinations show similar
therapeutic effectiveness, meaning that a plateau has been
reached with conventional chemotherapy. Moreover, relapses
following first line chemotherapy are constant and treatment
is associated with severe toxicity. New therapeutic approaches
with improved efficacy and better tolerability are therefore
urgently needed for NSCLC and other chemoresistant tumors.
In May, 2003, the US Food and Drug Administration approved
gefitinib for the treatment of patients with advanced NSCLC
previously treated with chemotherapy. This approval was based
on results of a phase II clinical study in which 142 patients
with refractory disease showed a 10% response rate to
gefitinib. The approval of the drug was granted despite
negative results from two randomized controlled trials in over
2000 previously untreated NSCLC patients, which showed no
benefit in survival, tumor response, or time to progression
when gefitinib was added to chemotherapy. Among other possible
reasons, the failure to demonstrate a benefit might have been
an important dilution of the benefit in a small cohort of
patients with tumors sensitive to EGFR inhibition by a larger
cohort of patients with insensitive tumors.
Preclinical studies are therefore needed to identify and
validate predictive factors to select patients with disease
likely to respond to EGFR inhibitors. Findings from phase II
clinical studies usually do not show a correlation between the
degree of EGFR expression and response to therapy.
The inventors have looked for genes that could be involved in
the response of human tumors to the growth inhibitory effect
of a competitive and reversible inhibitor of EGFR.
Growth-inhibition studies conducted in vivo with several human
tumor xenografts and in vitro with human HNSCC cell lines,
identified tumors responsive and tumors refractory to said
EGFR inhibitor. Genes whose expression levels were highly
correlated with response to said inhibitor were identified.
Expression ratio thereof was associated with resistance to
said inhibitor.
In support of this finding, the inventors have found that a
treatment combining RTK inhibitor activity and an inhibitor of
MKP-1 activity has a synergistic anti-tumor effect on RTK
inhibitor -resistant tumors.
An object of the invention is then to provide markers and
methods for predicting tumor response to RTK activity
inhibition.
Another object of the invention is to provide means for
potentiating the anti-tumor effect of RTK activity inhibitors.
The invention thus relates to tumor markers predictive of the
tumor cells resistance to inhibitors of RTK activity
consisting of phosphatase/kinase expression ratio as measured
in a tumor sample.
Such an expression level ratio appears indeed to be highly
correlated with the response to inhitors of RTK. A high
expression ratio was shown to be associated with resistance to
RTK inhibitor.
The invention particularly relates to markers consisting of
MKP-1/MKK7 mRNA ratio.
As shown in the examples, a high amount of MKP-1 (which codes
for a DSP which inactivates JNK) relative to MKK7 (which codes
for a kinase that is a positive upstream regulator of JNK) is
indicative of effective inhibition of JNK activity by MKP-1.
Such a ratio constitutes a positive index of JNK signaling
inhibition.
In other embodiments of the invention, the following mRNA
levels ratios may be used to assess the activity level of the
corresponding MAP kinases (sequences correspond to GenBank
accession numbers):
For the activation level of JNK1/2/3 (seq. NM 002750, NM
139046, NM 139047, NM 139049, NM 002752, NM 139068, NM 139069,
NM 139070, NM 002753, NM 138980, NM 138981, NM 138982), the
mRNA ratios of hVH2 (seq. NM 001394, NM 057158) and/or MKP7
(seq. NM 030640) and/or MKP5 (seq. NM 007207, NM 144728, NM
144729) and/or hVH1 (seq. NM 004417) and/or hVH5 (seq. NM
004420) to MKK7 (seq. NM 145185) and/or MKK4 (seq. NM 003010)
and/or CDC 42 (seq. NM 001791, NM 044472) and/or MAP4K3 (seq.
NM 003618). For the activation level of MAPK11 (seq. NM 002751, NM 138993)
and of MAPK14 (seq. NM 001315, NM 139012, NM 139013, NM
139014) and of MAPK12 (seq; NM 002969), the mRNA ratio of hVH2
(seq. NM 001394, NM 057158) and/or MKP7 (seq. NM 030640)
and/or MKP5 (seq. NM 007207, NM 144728, NM 144729) and/or hVH1
(seq. NM 004417) and/or hVH5 (seq. NM 004420) to MKK6 (seq. NM
002758, NM 031988) and/or MKK4 (seq. NM 003010).
The invention also relates to a method for predicting tumor
response to RTK inhibition, comprising measuring
phosphatase/kinase mRNA level in tumors.
The inventors have found that the degree of activation of
downstream kinase and the sensitivity of malignant tumors to
inhibition of RTK activity can be predicted with the mRNA
level of said genes involved in said signaling pathways.
In a preferred embodiment, said method comprises the
measurement of MKP-1/MKK7 mRNA ratio.
In other embodiments, said method comprises the measurement of
mRNA levels ratios such as above defined to assess the
activity level of the specific MAP kinases.
Advantageously, said expression level is measured by using a
real-time quantitative assay and calculating the relative mRNA
levels by the comparative Ct method following validation by
titration curve analysis (according to Applied Biosystems'
recommendations)
Oligonucleotides primers specific for the MKP-1 and MKK7
cDNA sequences are for example:
For MKP-1:
For MKK7
The invention also relates to the use of said tumor markers to
select patients with tumors susceptible to respond to RTK
inhibitors.
Advantageously, such a use further comprises administering to
a patient an effective amount of a RTK inhibitor in
combination with an inhibitor of phosphatase activity.
The treatment combining downstream MAP kinase inactivation,
via RTK inhibition, with phosphatase inactivation resulting
either spontaneously from low constitutive phosphatase
expression, in case of RTK-inhibitor sensitive tumor, or from
inhibition of phosphatase in cells with a high
phosphatase/kinase mRNA ratio, in the case of RTK inhibitor-resistant
tumors treated with an inhibitor of phosphatase,
inhibits tumor growth in part through induction of apoptosis.
The results given in the examples show a synergistic induction
of apoptosis in cells treated by a combination of RTK-inhibitor
and a phosphatase-inhibitor.
The invention thus relates to a method of treatment combining
inhibition of a phosphatase activity with suppression of MAP
kinase. Such a method of treatment results in a synergistic
anti-tumor effect in tumors with a high phosphatase/kinase
mRNA ratio, and have a significant anti-tumor activity in
tumors that are resistant to either treatment alone.
Advantageously, such a combined treatment also allows a
reduction in the effective dose of RTK inhibitor, whose
toxicity often remains limiting in patients.
Such a method is particularly useful for treating cancers in
which RTK signaling plays an important role for cell
proliferation.
The invention is then also directed to pharmaceutical
compositions comprising an effective amount of a RTK inhibitor
in combination with an inhibitor of phosphatase activity.
In a preferred embodiment, the RTK inhitor is gefitinib. In
another preferred embodiment, the inhibitor of phosphatase is
an isothiocyonate, such as PEITC. In a more preferred
embodiment, the treatment comprises using in combination,
together or sequencially, gefitinib and PEITC.
The doses will be advantageously of 50-100% the MTD (Maximal
Tolerated Dose) for each agent. Each agent will administred at
repeated doses, for example alternatively, each day, during.5-10
days.
Other characteristics and advantages of the invention will be
disclosed hereinafter, with reference to the figures 1 to 5,
which respectively represent:
Figure 1: Result of experiments with radiolabelled cDNAs from
IC9 and SC131 NSCLC xenografts hybridized to MAP kinase
Superarray membranes, showing high expression of MKP-1 mRNA in
SC131 and its absence in IC9. Figure 2: Histogram showing the distribution of MKP-1/MKK7
mRNA level ratios in 4 human NSCLC xenografts and in 3 human
HNSCC cell lines. Figure 3-a:Effect of gefitinib and PEITC alone or combined on
the growth and/or survival of the CAL33 human HNSCC cell line
in vitro. Cell number and viability was measured by the MTT
assay after a 72h incubation with or without the compounds at
the indicated concentrations. Figure 3-b: Effect of gefitinib and PEITC alone or combined on
the growth and/or survival of the PC3 human HNSCC cell line in
vitro. Cell number and viability was measured by the MTT assay
after a 72h incubation with or without the compounds at the
indicated concentrations. Figure 4: Rates of apoptosis as measured by the percentage of
cells in sub-G1 by propidium iodide staining and FACS analysis
of human HEP-2 HNSCC cells treated or not in vitro for 24h
with the indicated compounds. Figure 5a: Effect of Gefitinib administered at 120mg/kg on the
growth of the human TEP NSCLC xenograft in nude mice. Figure 5b: Effect of Gefitinib administered at 40 or 120mg/kg
on the growth of the human IC14 NSCLC xenograft in nude mice. Figure 5c: Effect of Gefitinib administered at 120mg/kg on the
growth of the human IC9 NSCLC xenograft in nude mice. Figure 5d: Effect of Gefitinib administered at 120mg/kg on the
growth of the human SC131 NSCLC xenograft in nude mice. Figure 5e: Effect of Gefitinib and PEITC alone or in
combination on the growth of the human SC131 NSCLC xenograft
in nude mice. Figure 5f: Effect of Gefitinib and PEITC alone or in
combination on the growth of the human HEP-2 HNSCC xenograft
in nude mice.
Gene expression analysis
In vivo studies were conducted with several human NSCLC
xenografted in nude mice to assess the effect of gefitinib on
tumor growth (see results in section 3 below). Two tumors
differing by their sensitivity to gefitinib were selected for
further studies: SC131 is refractory, and IC9 is sensitive to
tumor growth inhibition by gefitinib.
Possible determinants of gefitinib sensitivity in these tumors
were searched for by hybridizing radiolabeled tumor cDNA to
macroarray membranes (Superarray, Tebu) spotted with 96 DNA
sequences corresponding to genes involved in MAP kinase
signaling.
Among few other differentially expressed genes, MKP-1 was
identified as a gene whose expression was high in the
gefitinib-resistant SC131 tumor, and undetectable in the
gefitinib-sensitive IC9 tumor (Fig.1)
MKP-1 codes for a DSP which inactivates JNK (see above). On
the other hand, the MKK7/JNKK2 gene coding for a kinase that
is a positive upstream regulator of JNK was expressed at
similar levels in both tumors.
A real-time quantitative RT-PCR assay was then designed to
study the expression levels of MKP-1 mRNA in a larger panel of
tumors previously studied for their sensitivity to growth
inhibition by gefitinib either in vitro or in vivo (see
sections 2 and 3 below).
Since a high amount of MKP-1 relative to that of MKK7 is
likely to be indicative of effective inhibition of JNK
activity by MKP-1, the expression level of MKK7 mRNA was also
measured, and the ratio of MKP-1 to MKK7 mRNA was used as a
positive index of JNK signaling inhibition.
Oligonucleotide primers specific for the MKP-1 and MKK7 cDNA
sequences were chosen based on sequence alignments of the
known DSP and MKK7 family members cDNAs using the Clustal-X
software (Genebank accession no. XM_037430.6, NM_001395.1,
NM_004420.1, NM_001946.1, NM_004419.2, NM_001394.4,
NM_004090.2, NM_020185.3, NM_022076.2, NM_004418.2,
NM_080876.2, NM_152511.2, NM_080611.3, NM_152511.2,
NM_080611.3, NM_007026.1, NM_016364.2, NM_007240.1,
NM_003584.1, NM_007207.3, NM_004417.2, NM_005043.2,
NM_145185.1, NM_145329.1). PCR primers were designed using the
Primer-Express software (ABI, Les Ulys, France).
The sequences of the PCR primers used in this example are the
following: MKP-1, forward: 5'-GACTTCATAGACTCCATCAAGAATGC,
reverse: 5'-GGCAGTGGACAAACACCCTT; MKK7, forward: 5'-GGACCTGGATGTGGTGCTG,
reverse: 5'-GAAGACGTCCGTGTTGGTGA.
Real-time PCR was performed with a SYBR-green-based assay
(Eurogentec, Belgium) on a Gene-Amp 5700 Sequence Detector
System (ABI), and relative mRNA levels were calculated by the
comparative Ct method following validation by titration curve
analysis.
The data shown in Fig. 2 were obtained from four human NSCLC
xenografts and three human HNSCC cell lines characterized for
their response to gefitinib either in vivo or in vitro (see
sections 2 and 3 below). The two gefitinib-resistant tumors
(SC131 and Hep2) have a MKP-1/MKK7 ratio of 11 and 9
respectively, whereas the other gefitinib-sensitive tumors all
have a MKP-1/MKK7 ratio below 1.
These data show that a high MKP-1/MKK7 mRNA ratio correlates
with resistance to gefitinib treatment, suggesting that
effective suppression of JNK activity by high constitutive
MKP-1 levels protects tumor cells from growth inhibition
and/or apoptosis induced by EGFR inhibition.
In vitro studies of gefitinib sensitivity in three human
HNSCC cell lines
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) cell proliferation assay was used to determine
the growth inhibitory effect of gefitinib on the Hep2, CAL33
and PC3 human HNSCC cell lines. Cells were grown in 96-well
plates and incubated with varying concentrations of gefitinib
for 72h in DMEM medium supplemented with 10% foetal calf serum,
2 mM glutamine and antibiotics. Each measurement was done in
quadruplicate and the data were expressed as % proliferation
of control cells incubated in the absence of drug.
Phenylethyl isothiocyanate (PEITC) was used in addition to
gefitinib, a known JNK inducer that acts through suppression
of DUSPs activity, to look at the effect of MKP-1 inhibition
on gefitinib sensitivity (11). The IC50 (i.e. drug
concentration producing a 50% cell growth inhibition) of
gefitinib was first determined and PEITC used alone, and then
performed titration experiments with both drugs in combination
with a fixed gefitinib concentration (5x10-6M).
The Hep2 cell line showed a 4-fold increase in the IC50 for
gefitinib, compared with CAL33 and PC3, in agreement with
previously published studies given in Table 1 hereinafter:
Tumor cell lines | CAL33 | PC3 | HEP-2 |
IC50 gefitinib™ | 1,8×10-5 | 1,9x10-5 | 7,5x10-5 |
IC50PEITC | 1,8×10-4 | 4,9x10-4 | 3,5x10-4 |
Synergy | 0 | + | +++ |
In the combination study, a synergistic effect was obtained in
the PC3 cell lines, while no additive effect was obtained in
the CAL33 cell line (Fig. 3-a & 3-b).
Further studies concerned the effect of a 24h exposure to
gefitinib (40 µM) and PEITC (2 or 10 µM) alone or in
combination on the percentage of apoptotic cells in the
gefitinib-resistant Hep2 cell line, as measured by FACS
analysis of the % of cells in sub-G1 after propidium iodide
staining (Fig. 4).
The data show that a strong synergistic anti-proliferative
effect of the gefitinib/PEITC combination was obtained in
cells with a high MKP-1/MKK7 mRNA ratio, and that a
synergistic pro-apoptotic was obtained in gefitinib-resistant
cells with a strong imbalance of the MKP-1/MKK7 ratio when
gefitinib was combined with an inhibitor of DUSP activity.
In vivo studies of human tumor xenografts
The effect of gefitinib and PEITC alone or in combination on
the growth of human tumor xenografts in nude mice was tested
according to previously published procedures.
Briefly, tumor xenografts were maintained by successive s.c.
transplantation of tumor fragments in 8-week old nude mice.
Tumor growth curves were obtained by plotting the mean
relative tumor volume (i.e. the volume at a given time divided
by the volume at the start of the experiment) from groups of
at least 5 mice against time. Gefitinib was administered per
os at 40 or 120 mg/kg 5 days a week for 3 to 5 weeks, while
PEITC dissolved in corn oil was administered per os at 20 to
90 mg/kg every 2 days. No toxicity was observed in treated
mice under experimental conditions, as judged by the absence
of weight loss and clinical observation.
Three out of four NSCLC xenografts tested responded to
gefitinib at the dose of 120 mg/kg and to a lesser extent at
the dose of 40 mg/kg (Fig. 5-a, b & c). The SC131 NSCLC
xenograft did not respond to gefitinib (Fig. 5-d).
In contrast, a significant inhibition of tumor growth was
obtained with the combination of gefitinib (40 mg/kg) and
PEITC (20-90 mg/kg) in the SC131 NSCLC and the Hep2 HNSCC
xenografts (Fig. 5-e & f). With these two tumors, neither
gefitinib or PEITC had any antitumor effect when given alone.
These data show that DUSP inhibition by PEITC combined with
EGFR inhibition by gefitinib has a synergistic antitumor
activity in vivo in gefitinib-resistant tumors.
Annex to the application documents - subsequently filed sequences listing